New method for rapid and sensitive quantification of sulphide-producing bacteria in fish from arctic and temperate waters

The offensive, fishy, rotten H2S-off-odours in spoiled, aerobically and cold stored fish from arctic and temperate waters are generally caused by sulphide-producing bacteria (SPB), mainly Shewanella putrefaciens. In the present work, a new, rapid, simple and accurate method for estimation of the SPB content in fish from these areas is described. The quantification is based on the formation rate of iron sulphide during growth of SPBs incubated at 30 degrees C in a liquid growth medium containing cysteine, sodium thiosulphate and iron(III)citrate as specific substrates for iron sulphide formation. The iron sulphide turns the medium grey and masks the background fluorescence in the medium when the SPB content in the assay is approximately 10(9) cfu/ml. The fluorescence change could be detected instrumentally and the colour change visually. The method was developed and evaluated in tests with S. putrefaciens CCUG 13452 DT as well as naturally occurring SPBs in cod, salmon, wolf fish and coal fish. A linear correlation between the SPB count and detection time was obtained over the entire range from 1 to 10(9) cfu SPB/g, corresponding to detection times 17 and 1 h, respectively. The correlation is described by the equation: log cfu/g fish= -0.59(+/- 0.17) x DT+ 9.65(+/- 0.09), where DT is the detection time in hours. The model was valid for all the tested fish species and all tested naturally occurring SPBs in these species. The regression coefficients (R2) for cod, coal fish, wolf fish and salmon were 0.99, 0.92, 0.97 and 0.97, respectively. The detection level of the method is 1 SPB per sample tube, corresponding to 16 cfu/g fish. The method could be used to predict the remaining shelf life of the fish for different markets, even when the time-temperature history during storage of the fish is unknown.